Enhanced recovery of severely heat-injured, thermotolerant listeria monocytogenes from usda and fda primary enrichment media using a novel, simple, strictly anaerobic method1

A novel, simple, strictly anaerobic method was developed, which dramatically enhanced the recovery of severely heat-injured Listeria monocytogenes from pasteurized milk, compared to current aerobic methods. Cells of L. monocytogenes were grown in trypticase soy broth supplemented with 0.6% yeast extract (TSB-YE) at 43°C for 18 h and then severely heat-injured at 62.8°C for 5 min in raw milk in sealed, totally submerged thermal-death-time tubes. Recovery in primary enrichment media was enhanced in all cases by purging the headspace with N2 gas. The following treatments (in order of effectiveness) significantly improved recovery compared to conventional aerobic controls: addition of filter-sterilized cysteine + N₂ purging > pre-reduced Hungate media + N₂ purging = Oxyrase®+ lactate + N₂ purging > filter-sterilized cysteine - N₂ purging > Oxyrase®- lactate + N₂ purging. Addition of filter-sterilized cysteine to a final concentration of 0.5 g/1 with subsequent N2 purging increased recovery from 0% to 60% with the U.S. Department of Agriculture (USDA) UVM broth and from 11 to 100% with the Food and Drug Administration (FDA) LEB. Addition of either 1% pyruvate or a 10-fold higher concentration of cysteine (5.0 g/L) completely inhibited recovery. Faster and more efficient recovery of L monocytogenes using cysteine and N2 purging was attributed to the absence of oxidative stress during primary enrichment. Potential difficulties in recovering very severely injured cells are discussed. Copyright